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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B1/00—Instruments for performing medical examinations of the interior of cavities or tubes of the body by visual or photographical inspection, e.g. endoscopes; Illuminating arrangements therefor
  • A61B1/00142—Instruments for performing medical examinations of the interior of cavities or tubes of the body by visual or photographical inspection, e.g. endoscopes; Illuminating arrangements therefor with means for preventing contamination, e.g. by using a sanitary sheath
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
  • C12N1/16—Yeasts; Culture media therefor
  • C12N1/165—Yeast isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
  • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
  • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/645—Fungi ; Processes using fungi
  • C12R2001/72—Candida
  • C12R2001/74—Candida tropicalis

Definitions

• the present invention relates to a novel Candida tropicalis CJ-FID and a method for producing xylitol using the same. More particularly, the present invention relates to a novel Candida tropicalis CJ-FID derived from honey and a method for producing high purity xylitol with high yield and productivity, which includes culturing the Candida tropicalis CJ-FID itself or an alginate-immobilized Candida tropicalis CJ-FID in a fermentation medium containing high concentration xylose and sucrose to obtain a xylitol-containing culture, followed by purification with an activated charcoal column and an anion column, to finally obtain powdery xylitol.
• the xylitol productionusingmicroorganism has advantages in that separation of xylitol fromxylose is not required due to complete consumption of xylose after fermentation and mild conditions such as room temperature and atmospheric pressure are used.
• it is difficult to efficiently and selectively remove byproducts such as organic acids and glycerol, medium ingredients, and substances derived from microorganism, which are generally contained in a fermentation medium purification of high purity xylitol is difficult, resulting in low yield.
• Korean Patent Laid-Open Publication No. 10-2001-49918 discloses a method for producing xylitol from arabitol using Gluconobacter oxydans ATCC 621.
• xylitol is purified from 200 ml of a 45 g/L xylitol solution finally produced.
• this document does not disclose anaccurate purificationyieldresult .
• xylitol is produced from a low concentration (50g/L) xylitol solution, high concentration and efficiency xylitol production is not ensured, which renders mass production difficult.
• many studies of separation of a new yeast strain and a xylitol production method using the new yeast strain have been done.
• the present invention provides a novel Candida tropicalis CJ-FID.
• the present invention also provides a method for producing high purity xylitol with high yield and productivity.
• a xylitol-producing Candida tropicalis CJ-FID (KCTC 10457BP) .
• an alginate bead prepared by adding Candida tropicalis to an aqueous alginic acid solution with stirring to obtain a uniform solution and dripping the uniform solution into a calcium salt.
• yeast strains with excellent sugar tolerance and xylitol productivity under the above growth conditions were selected and their morphological and physiological characteristics were analyzed.
• the yeast strains have a nucleotide sequence as set forth in SEQ ID NO: 1.
• the yeast strains were designated as Candida tropicalis CJ-FID and deposited at Gene Bank of Korea Research Institute of Bioscience and Biotechnology (Accession No. : KCTC 10457BP) .
• the selected yeast strains were inoculated onto a 50 mL seedmedium containing 20 g/L of xylose , 5 g/L of yeast extract , 5 g/L of peptone, and 2 g/L of sucrose, in a 250 mL flask, and then shaking culture was performed at 200 rpm and 30 ° Cfor 12 hours.
• 5% of the seed culture was inoculated onto a 100 mLmainmediumcontaininglOOg/Lof xylose, 5 g/L of yeast extract , 5g/L of peptone, and lOg/L of sucrose, in a 500 mL flask, and then shaking culture was performed at 150 rpm and 30°Cfor 42 hours. Culture samples were taken from the resultant culture at predetermined intervals and then xylitol production in each culture sample was determined.
• xylose is contained in the fermentation medium in an amount of 50 to 200 g/L, and more preferably in an amount of 80 to 150 g/L. If the content of xylose is less than 50 g/L, the yield of xylitol may be lowered. On the other hand, if it exceeds 200 g/L, aproductivityenhancement maybe insignificant .
• the fermentation medium may be supplemented with 0.1-5 g/L of potassium phosphate (KH 2 P0 4 ) and 0.5-5 g/L of magnesium sulfate (MgS0 4 ) .
• Thexylitol-containing fractions were concentrated to more than 800 g/L in a concentrator and crystallized at 4 ° Q followed by addition of an alcoholic ethanol with stirring, to give fine xylitol crystals .
• the xylitol crystals were filtered in vacuum to partially remove ethanol and water. Finally, residual ethanol and water were removed by a vacuum dryer to give xylitol powders .
• FIG. 6 is a graph that illustrates xylitol productivity and yield with fermentation time of alginate-immobilized yeast cells in a 2L fermenter set to optimal fermentation conditions.
• FIG. 7 is a graph that illustrates xylitol productivity and yield with fermentation time of alginate-immobilized yeast cells in a 17 L fermenter set to optimal fermentation conditions .
• FIG. 8 is a graph that illustrates a change in xylitol concentration for xylitol-containing fractions obtained by application of a xylitol-containing culture to an activated charcoal column.
• FIG. 9 is a graph that illustrates a change in xylitol concentration for the xylitol-containing fractions obtained by application of the fractions of FIG.
• Example 1 Xylitol productivity with respect to initial xylose concentration After performing a seed culture as described above, the seed culture was inoculated onto a 10 L xylitol-producing main medium containing 0.5 g/L of yeast extract, 5 g/L of peptone, 10-30 g/L of sucrose, and 100-300 g/L of xylose, and cultured ina 15 L fermenter (Korea Fermentation Co . , Ltd.) . Fermentation conditions were as follows: stirring speed of 250 rpm, aeration flow speed of 1.0 wm, culture temperature of 30 ° C Xylitol productivity with respect to initial xylose concentration was measured and the results are shown in FIG. 1.
• the maximumxylitol production was 187g/L. At this time, the production yield of xylitol was excellent by 93.5%. It can be seen that addition of sucrose increased the yield of xylitol .
• Example 3 Sugar tolerance test for conventional xylitol-producing strains and Candida tropicalis CJ-FID (KCTC 10457BP) The Candida tropicalis CJ-FID according to the present invention and conventional xylitol-producing strains were evaluated for sugar tolerance and the evaluation results are presented in Table 1 below.
• the Candida tropicalis CJ-FID (KCTC 10457BP) of the present invention exhibited excellent xylose tolerance, as compared to currently known yeast strains . This means that the Candida tropicalis CJ-FID (KCTC 10457BP) of the present invention can produce xylitol in large scale in a high-concentration xylose-containing medium.
• Example 4 Evaluationof xylitol productivitywithrespect to density of alginate-immobilized cells To evaluatexylitol productivitywithrespect to the density of alginate bead-entrapped Candida tropicalis CJ-FIDyeast cells, after performing a seed culture as described above, alginate beads were prepared by mixing an alginate solution with the seed culture with varying the cell density (10 to 100 (OD value) ) .
• the alginate beads were inoculated onto 10 L xylitol-producing main media containing 5 g/L of yeast extract, 5 g/L of peptone, and 100 g/L of xylose, and cultured in a 15 L fermenter (Korea Fermentation Co., Ltd.) . Fermentation conditions were as follows: stirring speed of 250 rpm, aeration flow speed of 1.0 wm, culture temperature of 30 ° C. Xylitol productivity with respect to initial cell density was measured and the results are shown in FIG. 3. As shown in FIG. 3, when the cell density was 10 to 20 (OD value), xylitol productivity was about 0.5g/L.H. At the cell density of more than 50 (OD value) , xylitol productivity was more than 1.25g/L.H. At the cell density of 100 (OD value), xylitol productivity was maximal as 1.65g/L.H.
• Example 4 were inoculated with varying mass (2.5 to 50 g) onto xylitol -producing fermentation media in a 2 L fermenter and cultured under the same fermentation conditions as in Example 4. Xylitol productivity with respect to mass of the alginate beads was measured and the results are shown in FIG. 4. As shown in FIG. 4, xylitol productivity was maintained constant regardless of change in mass of the alginate beads.
• Example 7 Evaluation of xylitol productivity and yield with respect to repetitive use of immobilized cells Alginate bead-immobilized Candida tropicalis CJ-FID cells were prepared and fermented in the same manner as in Example
• the Candida tropicalis CJ-FID cells exhibited a five-fold increase for xylitol productivity and a more than 45% increase for xylitol yield, as compared to the Candida guilliermondii FTI 20037 cells.
• Example 8 Xylitol productivity with respect to fermentation time Alginate bead-immobilized Candida tropicalis CJ-FID cells were prepared and fermented in 2 L and 17 L fermenters in the same manner as in Example 4. Xylitol productivity was measured by varying the fermentation time and the results are shown in FIGS. 6 and 7. As shown in FIGS. 6 and 7, xylitol concentration was increased from 6 hours of the fermentation. With respect to the fermentation in the 2L fermenter (FIG. 6) , xylitol productivity was maximal at 24 hours of the fermentation. With respect to the fermentation in the 17 L fermenter, xylitol productivity was maximal at 36 hours of the fermentation.
• Example 9 Purification of xylitol fermentation culture Fractions of a xylitol fermentation culture that had passed through an activated charcoal column and an anion column were measured -for xylitol concentrations. The results are shown in
• FIGS. 8 and 9. The fractions of FIG. 9 were analyzed by high-performance liquid chromatography (HPLC) and the results are shown in FIG. 10.
• the fractions of FIG. 9 were concentrated to different concentrations and then xylitol crystallization yields with respect to the concentrations were measured. The results are shown in FIG. 11.
• a fraction 4 exhibited the highest xylitol concentration.
• fractions 3 through 5 exhibited the highest xylitol concentration. From the chromatogram of FIG.
• the fractions that had passed through the activated charcoal column and the anion column contained highly purified xylitol.
• the purified xylitol solutions were crystallized to different concentrations . As the concentration of the xylitol solutions increased, the xylitol crystallization yield increased proportionally.
• Example 10 Xylitol concentration and yield according to xylitol fermentation and purification Xylitol concentrations and yields measured after the fermentation and the purification are summarized in Table 3 below.

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Citations (4)

• 2004
  • 2004-05-13 KR KR1020040033733A patent/KR20050025059A/ko not_active Application Discontinuation
  • 2004-05-18 EP EP04733702A patent/EP1651753B1/en not_active Expired - Lifetime
  • 2004-05-18 WO PCT/KR2004/001172 patent/WO2005010171A1/en active Application Filing
  • 2004-05-18 PL PL04733702T patent/PL1651753T3/pl unknown
  • 2004-05-18 JP JP2006520989A patent/JP4365862B2/ja not_active Expired - Lifetime
  • 2004-05-18 DE DE602004031850T patent/DE602004031850D1/de not_active Expired - Lifetime
  • 2004-05-18 BR BRPI0412250A patent/BRPI0412250B8/pt active IP Right Grant
  • 2004-05-18 US US10/564,274 patent/US20060110811A1/en not_active Abandoned
  • 2004-05-18 AT AT04733702T patent/ATE502101T1/de active
  • 2004-05-18 CN CNA2004800215489A patent/CN1829790A/zh not_active Withdrawn

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